Systemic rheumatic diseases are characterized by the occurrence of circulating autoantibodies to defined intracellular targets (reviewed in von Mühlen and Tan, 1995). Among the earliest of those autoantibodies to be identified were the anti-Sm, which are closely associated with systemic lupus erythrematosus (SLE) (Tan and Kunkel 1966). Thus, anti-Sm antibodies have been included as one of the American College of Rheumatology classification criteria for this disease (Tan et al., 1982). Apart from autoantibodies targeting the Sm-complex anti-DNA, anti-PCNA, anti-U1-RNP, anti-nucleosome, anti-histone, anti-Ro/SS-A, anti-La/SS-B, anti-ribosomal RNP and anti-phopholipid antibodies are frequently found in patients suffering from SLE (von Mühlen and Tan 1995).
In average anti-Sm reactivity is found in 5-30% of patients with SLE, although the specific frequency will vary depending on the detection system and the ethnicity of the SLE population (Abuaf et al., 1990; Jaekel et al., 2001). The Sm-antigen is part of the spliceosomal complex that catalyzes the splicing of nuclear pre-mRNA (Seraphin, 1995; Lerner et al., 1980). The complex itself comprises at least nine different polypeptides with molecular weights ranging from 9-29.5 kDa [B (B1, 28 kDa), B′(B2, 29 kDa), N (B3, 29.5 kDa), D1 (16 kDa), D2 (16.5 kDa), D3 (18 kDa), E (12 kDa), F (11 kDa) and G (9 kDa)] (Hoch, 1994). All of those core proteins can serve as targets of the anti-Sm immune response, most frequently the B and D polypeptides, which are therefore considered the major antigens (Hoch, 1994; Brahms et al., 1997; Ou et al., 1997). However, SmBB′ and U1 specific RNPs which are frequently the target of autoantibodies in patients with MCTD share crossreactive epitopes, consequently SmD is regarded as the most specific Sm-antigen (van Venrooij et al., 1991; Hoch et al., 1999). Within the SmD family the SmD1/D3 pattern is at least four times more common than SmD1/D2/D3 recognition with a pronounced immunoreactivity to SmD1 (Hoch et al., 1999). In epitope-mapping studies, several linear and conformational epitopes have been mapped on the SmB- and D-proteins (Rokeach et al., 1992; Hirakata et al., 1993). On SmD1 and BB′ the major reactivity was predominantly found in the C-terminal extensions (Rokeach et al., 1992; Hirakata et al., 1993; Rokeach and Hoch, 1992). The epitope PPPGMRPP (SEQ ID NO: 2) that occurs three times within the C-terminal extensions of SmBB′ was shown to crossreact with other prolin rich structures of spliceosomal autoantigens such as the U1 specific antigens and of retroviral proteins such as p24 gag of HIV-1 (De Keyser et al., 1992). Follow-up studies and immunization experiments revealed that this motif is consistently the earliest detectable SmBB′ epitope acting as starting point of epitope-spreading events within the BB′ molecule and to the SmD-polypeptides (Arbuckle, 1999; Greidinger and Hoffman, 2001).
A recent study identified five linear epitopes on SmD2 and four on SmD3 distributed on the entire molecules (McClain et al., 2002). All of these epitopes share basic properties and are exposed on the surface of the protein rendering them antigenic (McClain et al., 2002).
One of the described B-cell epitopes on SmD3 (epitope 4; aa 104-126) displayed close homology to an antigenic region from the SmD1 protein finally leading to crossreactivity (McClain et al., 2002). For diagnostic purposes a synthetic peptide corresponding to the C-terminal extension of SmD1 was used to develop an ELISA system with diagnostic sensitivities and specificities ranging from 36-70% and from 91.7% and 97.2%, respectively (Riemekasten et al., 1998; Jaekel et al., 2001). Recently, it has been shown, that the polypeptides D1, D3 and BB′ contain symmetrical dimethylarginine (referred to herein as sdR or sDMA) constituting a major autoepitope within the C-terminus of SmD1 (Brahms et al., 2000; Brahms et al., 2001). In one of these studies a synthetic peptide of SmD1 (aa 95-119) containing sDMA demonstrated significant increased immunoreactivity compared to the non-modified peptide reflecting a conflict to previous data (Riemekasten et al., 1998; Brahms et al., 2000).
In WO 99/11667 a method is described for producing peptides containing methylated arginines and that constitute immunogenic determinants of antibodies present in sera from patients with SLE or Epstein-Barr virus (EBV) and wherein the methylation is a prerequisite for reacting with said antibodies. However, these peptides are generally described and no connection between peptide sequence and ability to diagnose autoimmune disease has been disclosed.